Many attempts have been recently focussed on the development of procedures for the targeted release of pharmaceutical agents in specific sites in a patient or to obtain a slow release of drugs in the patient. It is known that the effectiveness of a drug can increase when the appropriate target site is effectively reached by the drug, or when the drug is preferably released in the organ to be treated or in the cell to be treated. Furthermore, the toxicity of a drug can be reduced when the total amount of administered drug is minimised although maintaining its therapeutical action upon slow release of the drug. The interest in drug administration systems relates both to conventional agents, many of which are relatively simple organic molecules, and to more complex pharmacologically active agents such as peptides, proteins, enzymes, antibodies, antisense oligonucleotides, decoy oligonucleotides, cytokines, nucleic acids, and combinations thereof, etc.
A field of recent interest relates to the use of red blood cells (hereinafter designated as “erythrocytes” or “RBCs”) as carriers to release therapeutical dosages of drugs in the blood circulation in low doses or at a desired site in a patient. The erythrocytes may be “loaded” with biologically active agents and by means of a process in which the cell membranes of the erythrocytes are made permeable and one or more agents are added to the erythrocytes then resealing the cell membranes. These “loaded” or “treated” erythrocytes offer several advantages as drug release systems and targeting systems as they are biodegradable, can be maintained in the circulation for long periods of time and can be targeted to cells such as for example macrophages.
Processes for the preparation of a cell suspension loaded in a physiological solution are disclosed in German patent No. 23 26 244 and in German patent applications published with No. (0S) 23 26 161 and 24 95 119, in which the cell membranes of erythrocytes are lysed by osmotic pressure and an electric field, respectively.
The paper “Erythrocytes as carriers of primaquine-preparation: characterization and evolution” (Naresh Talwar et al.; Journal of Controlled Realease, 20 (1992) 133-142) discloses the encapsulation of phosphate in erythrocytes. It is indicated that the suggested method involves the lysis of erythrocytes and that the treated erythrocytes are washed. However, it is nowhere mentioned or suggested that the concentration of the treated erythrocytes must and/or can be increased.
U.S. Pat. No. 4,224,313, (Zimmermann et al), discloses a method to prepare a mass of cells loaded in suspension in a solution increasing the permeability of the cell membrane by an externally induced osmotic pressure or an electric field or both. The material to be loaded includes a pharmaceutical agent which has the ability, when incorporated in a cell, to prematurely destroy the cell membranes, and a stabilising agent that can inhibit the reaction of the pharmaceutical agent with the cell membranes.
U.S. Pat. No. 4,478,824 (Franco et al.) discloses a method for incorporating substances within erythrocytes changing the internal osmotic pressure of RBCs by means of the action of chemical agents, such as DMSO and glycerol, which can cross the cell membrane and enter the cells by diffusion.
U.S. Pat. No. 4,652,449, (Ropars et al.), discloses a method and an apparatus to incorporate materials within erythrocytes by osmotic pressure. The method and the apparatus have been employed and tested only on large volumes of blood. This limits many applications to employing autologous blood, i.e. blood obtained from the same patient which will then receive the blood loaded with the drug.
U.S. Pat. No. 4,931,276, (Franco et al.), discloses a method for encapsulating non-ionic agents in erythrocytes. The method has a limited effectiveness when the desired agent to be incorporated is not anionic, or is anionic or polyanionic but is not present in the virtually isotonic aqueous means at a concentration sufficient to cause the required increases in the permeability of the cells without the destruction of the cells.
Heubsch et al., J. Cell. Physiol., 122:266-272 (1985), show that, in osmotically swollen cells, the double lipid layer that forms the membrane detaches from the cytoskeleton of the cell, and the cell considerably varies its size and becomes spherical. This does not occur in normal conditions.
The patent application with publication number EP1466968 discloses a method and a machine for encapsulating active substances in erythrocytes.
Reviews of methods for incorporating substances in cells are given by Franco et al. in Life Science 32:2763-2768 (1983), Am. J. Hematol. 17:393-400 (1984), e J. Cell. Physiol. 129:221-229 (1986).
Although the use of erythrocytes as drug release systems has been investigated by many, the methods and the devices which implement these methodologies have not yet been developed to the point of being applied normally in clinical practice, in diagnostics and in research.
Furthermore, the methodologies and the devices developed up to now are not sufficiently flexible as to allow to obtain erythrocytes for any kind of use in the therapeutic, diagnostic and research field. In particular, by following the procedures disclosed in the state of the art, it is often not possible to obtain a product that can actually be used in the diagnostic (or therapeutic) field.
A recent example of an apparatus for encapsulating a compound in erythrocytes is also disclosed in patent U.S. Pat. No. 6,139,836. Although this apparatus represents a considerable improvement with respect to the previous state of the art, it is relatively complex, expensive and difficult to use. In this connection, it should be noted that the operation of the apparatus of patent U.S. Pat. No. 6,139,836 requires the presence and the continuous intervention of a specialised operator which must operate the different components of the apparatus in the correct sequence. Therefore, the treatment (loading) of erythrocytes is relatively time-consuming and involves the risk of the operator making mistakes.
The known apparatuses cannot be appropriately carried. Due to the fact they cannot be carried, the procedure needs to therefore be carried out in dedicated structures and it is not possible to operate at sites which are more accessible to patients. Furthermore, the known apparatuses require the continuous intervention of specialised staff and are not always precise and/or sufficiently effective.
It is an object of the present invention to provide an apparatus, a kit, a use and a method, which allow to overcome at least partially the drawbacks of the state of the art and are at the same time easy and cost-effective to implement.